andes virus nucleocapsid protein interrupts pkr dimerization to
TRANSCRIPT
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Andes Virus Nucleocapsid Protein Interrupts PKR Dimerization to Counteract The Host 1
Interference in Viral Protein Synthesis. 2
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Zekun Wang and Mohammad A Mir# 4
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Department of Microbiology, Molecular Genetics and Immunology, University of Kansas 6
Medical Center, Kansas City, Kansas, USA. 7
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#To whom correspondence should be addressed: Mohammad A Mir E-mail: 9
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Running Title: Andes Virus Nucleocapsid Protein Inhibits PKR Dimerization. 12
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JVI Accepts, published online ahead of print on 19 November 2014J. Virol. doi:10.1128/JVI.02347-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 20
Pathogenic hantaviruses delay type I interferon response during early stages of viral infection. 21
However, the robust interferon response and induction of interferon stimulated genes, observed 22
during later stages of hantavirus infection fails to combat the virus replication in infected cells. 23
Protein kinase R, a classical interferon stimulated gene product phosphorylates the eukaryotic 24
translation initiation factor eIF2α and causes translation shutdown to create roadblocks for the 25
synthesis of viral proteins. The PKR induced translation shutdown helps host cells to establish an 26
antiviral state to interrupt virus replication. However, hantavirus infected cells do not undergo 27
translation shutdown and fail to establish an antiviral state during the course of viral infection. 28
Here we show for the first time that Andes virus infection induced PKR over-expression. 29
However, the over-expressed PKR was not active due to significant inhibition of 30
autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited 31
PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The 32
studies reported here have established hantavirus nucleocapsid protein as a new PKR inhibitor. 33
These studies have provided mechanistic insights for hantavirus resistance to host interferon 34
response and have solved the puzzle for the lack of translation shutdown observed in hantavirus 35
infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective 36
evolutionary pressure on hantaviruses to evade PKR antiviral response for survival. We envision 37
that evasion of PKR antiviral response by NP has likely helped hantaviruses to exist during 38
evolution and survive in infected hosts having multifaceted antiviral defense. 39
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Importance 42
Protein kinase R, a versatile antiviral host factor shuts down the translation machinery upon 43
activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The 44
studies reported in this manuscript reveal that hantavirus nucleocapsid protein counteracts PKR 45
antiviral response by inhibiting PKR dimerization, required for its activation. We report the 46
discovery of a new PKR inhibitor whose expression in hantavirus infected cells prevents the 47
PKR induced host translation shutdown to ensure the continuous synthesis of viral proteins, 48
required for efficient virus replication. 49
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Introduction 62
Hantaviruses are segmented negative strand RNA viruses of the Bunyaviridae family. Their 63
genome is composed of three RNA segments S, L and M, encoding viral nucleocapsid protein 64
(NP), viral RNA dependent RNA polymerase (RdRp) and glycoprotein precursor (GPC) 65
respectively (1). The GPC is post-translationally cleaved at a conserved WAASA motif into two 66
glycoproteins Gn and Gc (2). Hantaviruses are carried by rodents. Humans are infected by the 67
inhalation of aerosolized excreta of infected rodent hosts. Their infections cause hemorrhagic 68
fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with 69
mortality rates of up to 12% and 50%, respectively in certain outbreaks (3). Annually, 150,000 70
to 200,000 cases of hantavirus infection are reported worldwide (4). There is no FDA approved 71
vaccine or an antiviral therapeutic against hantavirus infections. Hantaviruses usually do not 72
transmit from human to human. However, Andes virus (ANDV), a New World hantavirus 73
species has been reported to undergo human to human transmission (5). Hantaviruses primarily 74
target endothelial cells having the receptor (β3 integrin) for virus attachment and entry. Their 75
replication occurs exclusively in the host cell cytoplasm. Hantaviral RdRp initiates transcription 76
by a unique cap snatching mechanism to generate 5' capped viral mRNAs (6-8). Despite their 5' 77
caps, viral mRNAs have to actively compete with the host cell transcripts for the same 78
translation machinery. Our recently published findings suggest that hantaviruses use a novel NP-79
mediated translation initiation mechanism that lures the host translation apparatus for the 80
preferential translation of viral mRNA (9). 81
The endothelial cells (ECs) respond differently to pathogenic and nonpathogenic 82
hantavirus infection. Previous studies have shown that nonpathogenic Prospect Hill virus (PHV) 83
strongly stimulates the expression of interferon (IFN) and interferon stimulated genes (ISGs) 84
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during the early stage of viral infection that limits PHV replication in ECs (10, 11). In contrast, 85
pathogenic Hantaan Virus (HTNV), Sin Nombre virus (SNV), New York-1 virus (NY-1) and 86
ANDV induce very weak innate immune response during the early stages of infection. As a 87
result, pathogenic hantaviruses successfully replicate in ECs (10, 11). Moreover, both pathogenic 88
and nonpathogenic hantaviruses replicate to same titers in IFN deficient Vero E6 cells (10). 89
These observations suggest that pathogenic hantaviruses have evolved a strategy to delay the 90
early interferon induction for efficient replication in ECs. Further studies revealed that Gn 91
cytoplasmic tail domain inhibits IFN induction (12). Interestingly, both pathogenic and 92
nonpathogenic hantaviruses strongly induce the expression of both IFN and ISGs at later stages 93
of viral infection, which fails to combat pathogenic hantavirus replication (11). Moreover, 94
pathogenic hantaviruses are sensitive to IFN pretreatment or post-treatment within 12 hours of 95
virus infection. The IFN treatment 15 to 24 hours post virus infection induces ISG response, 96
which fails to combat virus replication (10, 13). These observations suggest that pathogenic 97
hantaviruses have evolved strategies to counteract antiviral effects of ISGs by an unknown 98
mechanism. One of the crucial ISGs implementing the antiviral actions of interferon is the 99
protein kinase R (PKR), a double stranded RNA activated protein kinase that phosphorylates and 100
inactivates the alpha subunit of eukaryotic translation initiation factor 2α (eIF2α) (14). The 101
phosphorylation of eIF2α inhibits translation initiation, which imposes restrictions on the 102
synthesis of viral proteins in the host cell. The PKR antiviral response promotes the 103
establishment of antiviral state in the host cell, aimed to limit the virus replication and 104
dissemination in the host (15). The hantavirus NP-mediated translation strategy is also sensitive 105
to PKR activation due to its dependence upon eIF2α. However, viruses have evolved numerous 106
strategies to counteract PKR antiviral response. This includes the expression of virus encoded 107
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decoy dsRNA, PKR degradation in virus infected cells, sequestration of viral dsRNA, inhibition 108
of PKR dimerization, dephosphorylation of downstream target eIF2α or production of PKR 109
pseudosubstrates (16-18). It is well understood that hantavirus infected cells do not undergo 110
translation shutdown even though the PKR is over-expressed due to virus induced IFN response 111
during later stages of infection. This led to the hypothesis that hantaviruses have evolved a 112
strategy to counteract the PKR induced translation shutdown in virus infected cells that ensures 113
continuous manufacture of viral proteins during the course of viral infection. In this study we 114
show that ANDV NP interference in PKR dimerization, a critical step for its activation. The 115
resulting PKR inactivation prevents translation shutdown in virus infected cells and facilitates 116
viral protein synthesis during the course of infection. 117
Materials and Methods 118
Cell culture and virus propagation: Human embryonic kidney 293T (HEK293T), African 119
green monkey kidney (Vero E6) and human hepatocarcinoma Huh7 cells were grown in DMEM 120
(HyClone) supplemented with 10% fetal bovine serum (HyClone), 2 mM L-glutamine, 100 U/ml 121
penicillin and 100 μg/ml streptomycin. Human umbilical vein endothelial cells (HUVEC) were 122
purchased from Lonza and cultured in EGM BulletKit medium (Lonza). Lentiviruses were 123
packaged in HEK293T cells. Briefly, the gene of interest was cloned in pLenti-CMV vector. The 124
resulting expression plasmid was co-transfected into HEK293T cells along with packaging 125
plasmid psPAX2 (Addgene 12260) and envelop plasmid pMD2.G (Addgene 12259). 126
Supernatants from transfected cells were harvested at 48 and 72 hours post-transfection. 127
Lentivirus particles were concentrated by ultracentrifugation and quantified by qPCR based 128
assay (19). ANDV (strain Chile-9717869) was propagated in Vero E6 cells. Briefly, ANDV was 129
inoculated in Vero E6 cells at an MOI of 0.03. The cells were cultured in DMEM containing 2.5% 130
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FBS. Supernatant from cultured cells was collected thirteen days post-infection. The viral titers 131
in the supernatant were determined by plaque assay (20). All work with infectious ANDV was 132
performed in biosafety level-3 laboratory at the University of Kansas Medical center. 133
Antibodies: The primary antibodies for PKR (cat. # 12297), eIF2α (cat. # 5324), phosphorylated 134
eIF2α (cat. # 9721) were from Cell Signaling Technologies. The primary antibody for 135
phosphorylated PKR (cat. # ab81303) was from Abcam. The primary antibodies for FLAG tag 136
(clone M2, cat. # F1804), β-actin (cat. # A5441) and GFP (cat. # G6795) were from Sigma. The 137
primary antibodies for c-Myc tag (clone 9E10, cat. # sc-40) and normal mouse IgG (cat. # sc-138
2025) were from Santa Cruz. The primary antibody for GAPDH (cat. # A01622) was from 139
GenScript. The rat anti-serum used for the detection of NP was from our lab. 140
Plasmids: Total RNA purified from ANDV infected Vero E6 cells was reverse transcribed using 141
random primers. The resulting cDNA was used to PCR amplify the ANDV NP open reading 142
frame (ORF). The ORF encoding SNV NP was similarly PCR amplified from pTSNV N vector 143
(21). The ORF encoding the nucleocapsid protein of hantaan virus (HTNV), strain M14626 was 144
synthesized by Integrated DNA technologies (IDT). Each ORF was inserted into the 145
pcDNA3.1(+) backbone that was previously modified to incorporate either Myc or FLAG tag at 146
the N-terminus of the ORF. The pHis-NP plasmid, expressing C-terminally His tagged ANDV 147
NP was constructed by the insertion of NP ORF into pTriEx1.1 backbone. The pMyc-NP (Δ175-148
230) and pHis-NP (Δ175-230) plasmids that express ANDV NP mutant lacking the RNA binding 149
domain were subcloned by overlapping PCR, as previously reported (22). The lentiviral vectors 150
expressing N-terminally Myc tagged wild type or mutant of NP were subcloned from pMyc-NP 151
or pMyc-NP (Δ175-230) plasmid into pLenti-CMV backbone (Addgene 17448). The pMyc-PKR 152
and pFLAG-PKR plasmids expressing Myc and FLAG tagged PKR, respectively, were 153
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constructed by inserting the PKR ORF into modified pcDNA3.1(+) backbone as mentioned 154
above. The PKR ORF was amplified from cDNA generated by the reverse transcription of an 155
RNA sample obtained from HUVECs pretreated with IFNα. All plasmids were sequenced to 156
verify sequence integrity. 157
Transfection and virus infection: Plasmid and polyinosinic-polycytidylic acid (poly I:C) 158
transfections were carried out using Turbofect transfection reagent (Thermo) and lipofectamine 159
2000 (invitrogen), respectively, following manufacturer’s instructions. The lentiviral infection 160
was carried out in biosafety level-2 environment. Briefly, polybrane (8.0 μg/ml) was added to 161
EBM media, followed by the addition of lentivirus of required MOI, using a high concentration 162
stock. The resulting mixture was added to HUVECs and incubated for twelve hours, and then 163
replaced with fresh media. Cells were harvested 48 hours post-infection. For lentivirus based 164
stable cell line generation, HEK293T cells were infected with lentivirus using the same method 165
as mentioned above, except the cells were selected with puromycin (3 μg/ml). ANDV infection 166
was carried out in biosafety level-3 environment. Briefly, ANDV from a high concentration 167
stock was diluted in DMEM containing 2.5% FBS to achieve required MOI. Cells were 168
incubated with diluted virus preparation for 1 hour with periodic rocking. Cells were rinsed 169
twice with PBS to remove the unabsorbed virus. Cells harvested at this time point were referred 170
as one hour post-infection and used as baseline for virus replication. The remaining cells were 171
allowed to grow in fresh medium and harvested at different time points post-infection. 172
Coimmunoprecipitation: HEK293T cells seeded in 6 cm dishes were cotransfected with 173
required plasmids. Cells were carefully washed once with PBS 48 hours post-transfection, 174
followed by lysis in 600 μl of NP-40 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% 175
NP-40, 10% glycerol, 1 mM EDTA), supplemented with protease and phosphatase inhibitor 176
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cocktails (Roche). Cell lysates were clarified by centrifugation at 16,000×g for 15 min at 4°C 177
and supernatants were collected. 50 μl of the supernatant was mixed with equal amount of 2× 178
SDS loading buffer and saved as input. The remaining supernatant was pre-cleared with protein 179
G agarose beads (invitrogen) for 30 min and incubated for 4 hours with 1 μg of required antibody. 180
The mixture was further incubated with 40 μl of protein G agarose beads by continuous rotation 181
for 1 hour at 4°C. The beads were pelleted down by a brief centrifugation and washed four times 182
with lysis buffer. The material bound to washed beads was eluted by boiling with 45 μl of 1× 183
SDS loading buffer. The boiled samples were briefly centrifuged and 15 μl of the supernatant 184
were examined by western blot analysis. 185
Western blot analysis: Cells were washed once with phosphate buffer saline (PBS) and lysed 186
with RIPA buffer, supplemented with protease inhibitor cocktail (Roche). Cell lysates were 187
mixed with equal volume of 2× SDS loading buffer and boiled at 95°C for 5 min. Samples were 188
separated on 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane 189
was blocked with 5% non-fat milk in PBST buffer (1× PBS, 0.05% Tween 20). The membrane 190
was incubated with primary antibody, followed by washing and further incubation with the 191
secondary antibody conjugated to HRP. The antibody concentrations were used as suggested by 192
the manufacturer. The protein signal was detected by chemiluminescence. 193
35S metabolic labeling: Cells grown in 6-well plates were washed twice with starvation media 194
(DMEM containing 10% FBS and deficient in methionine and cysteine) and cultured in the 195
starvation media for 30 min. to deplete intracellular pools of methionine and cysteine. Cells were 196
then incubated for 40 min. with 1 ml of starvation media containing 300 μCi 35S labeled 197
methionine and cysteine (PerkinElmer). Cells were washed once with PBS and lysed with 100 198
μl RIPA buffer. The cell lysates were mixed with equal volume of 2× SDS loading buffer and 199
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boiled at 95°C for 5 min. Protein samples were separated on 10% SDS-PAGE. The gel was 200
stained with coomassie brilliant blue dye, dried on filter paper and exposed to X-ray film over 201
night at -80°C. 202
Real time PCR: Total RNA was extracted from cells using RNeasy Kit (Qiagen), following the 203
manufacturer’s instructions. One μg of the purified RNA was reverse transcribed in a total 204
volume of 20 μl, using M-MLV Reverse Transcriptase (invitrogen) according to manufacture’s 205
protocol. The cDNA was diluted 10 fold and 5 μl of the diluted cDNA sample was used in a 20 206
μl real time PCR reaction for the quantitative estimation of mRNA of interest, using an 207
appropriate primer set. Each reaction was performed in triplicates. Similarly, the mRNA levels of 208
a housekeeping gene β-actin were quantified as an internal control. Real time PCR reactions 209
were performed on ABI 7500 real time PCR system (Applied Biosystems), using SYBR green 210
PCR master mix (Roche). We used relative quantification method for data analysis, as previously 211
mentioned (23). Fold change in mRNA levels and standard deviation shown as error bars were 212
calculated as previously reported (6). The primer pairs used for the quantitative estimation of 213
mRNA levels for PKR (5'- GCC GCT AAA CTT GCA TAT CTT CA -3' and 5'-TCA CAC GTA 214
GTA GCA AAA GAA CC -3') IFN-β (5'- GCT TGG ATT CCT ACA AAG AAG CA -3' and 5'- 215
ATA GAT GGT CAA TGC GGC GTC -3') and β-actin (5'- GAG CAC AGA GCC TCG CCT 216
TT- 3' and 5'- TCA TCA TCC ATG GTG AGC TGG- 3') were selected from primer bank and 217
have been previously verified for the use in real time PCR analysis (24). The primers used for the 218
quantitative estimation of ANDV S-segment RNA by real time PCR analysis were: 5'- CAG 219
CTC GTG ACT GCT CGG C-3' and 5'- GTA GAC ACA GCT GCC CGT CTA C -3'. These 220
primers have been verified for the use in real time PCR. 221
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Results 223
ANDV induces PKR over-expression but fails to shutdown the host translation machinery. 224
Pathogenic hantaviruses such as ANDV or SNV are well known to inhibit early interferon 225
(IFN) response after infection (11, 25, 26). However, the strong expression of both IFN and IFN 226
stimulated gens (ISGs) is observed in the later stages of their infection. Interestingly, the delayed 227
but strong interferon response does not inhibit virus replication (11). This observation is 228
consistent with the fact that pathogenic hantaviruses are sensitive to early IFN treatment in cell 229
culture. The IFN treatment remains ineffective beyond 15 hours post-viral infection (10). These 230
observations led to the hypothesis that pathogenic hantaviruses may have evolved strategies to 231
antagonize the antiviral effects of some ISGs. To test this hypothesis, we infected HUVECs with 232
ANDV and harvested the cells at different time points post-infection. The cells were examined 233
for PKR expression at both mRNA and protein levels. We observed that ANDV infection 234
induced PKR expression at transcriptional level (Fig. 1B). The PKR protein levels started rising 235
in virus infected cells from 24 hours post-infection and remained steadily high onwards (Fig. 1A). 236
PKR is one of the classical ISGs that promotes the establishment of antiviral state in virus 237
infected cells by shutting down the host cell translation machinery to create barriers for viral 238
protein synthesis. An examination of virus infected cell lysates by western blot analysis revealed 239
that PKR over-expression had no impact upon the endogenous steady state levels of ANDV NP 240
(Fig. 1A). This observation suggested that PKR over-expression may not impact the host 241
translation machinery. To test this hypothesis, we monitored the de novo protein synthesis in 242
virus infected HUVECs at different time points post infection, using 35S methionine and cystine 243
labeling, as mentioned in Materials and Methods. We did not observe any change in the rate of 244
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host protein synthesis due to virus infection, suggesting that ANDV has evolved strategies to 245
counteract PKR antiviral response (Fig. 1C and 1D). 246
ANDV regulates PKR activation. 247
We next asked how ANDV counteracts the over-expressed PKR antiviral response to 248
maintain the cellular translation machinery in the functional state. The PKR undergoes 249
dimerization and autophosphorylation for activation. The activated PKR then phosphorylates the 250
downstream target eIF2α to induce transient shutdown of host translation machinery. To 251
determine whether ANDV interferes in the activation of PKR-eIF2α pathway, we infected 252
HUVECs with either ANDV or treated them with either IFNα or IFNα along with poly I:C, as 253
positive controls. Cells were harvested at different time points post-infection. The expression of 254
phosphorylated PKR and eIF2α were examined by western blot. As expected, both positive 255
controls induced the PKR over-expression and phosphorylation of both PKR and eIF2α (Fig. 2A). 256
However, ANDV infection induced the PKR over-expression similar to positive control, but the 257
over-expressed PKR was poorly phosphorylated, leading to the poor phosphorylation of eIF2α 258
(Fig. 2A). This observation clearly demonstrates that ANDV has evolved strategies to inhibit the 259
PKR activation in virus infected cells. 260
It has been previously reported that lentivirus infection triggers IFN response that activates 261
PKR-eIF2α pathway (27-30). We next compared the activation of PKR-eIF2α pathway in 262
lentivirus and ANDV infected cells at two different time points post-infection. It is evident from 263
Fig. 2B that both lentivirus and ANDV infections induced PKR over-expression to the similar 264
level. However, the PKR-eIF2α pathway was poorly activated in ANDV infected cells as 265
compared to cells infected with lentivirus, evident from poor phosphorylation of both PKR and 266
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eIF2α (Fig. 2B). These results further support that ANDV has evolved strategies to inhibit PKR 267
activation. 268
To determine whether ANDV replication was sensitive to PKR activation, we asked 269
whether over-expression of PKR from a transfected plasmid would interfere with ANDV 270
replication. The expression and activation of PKR was tested in Huh7 cells either lacking or 271
harboring the replicating ANDV. Huh7 cells were used in this experiment due to their better 272
transfection efficiency and poor expression of TLR3 (31), which is important for hantavirus 273
recognition (32). Unlike HUVECs, the ANDV infection in Huh7 cells did not induce high PKR 274
expression (Fig. 2C, compare lanes 1 and 2), which was likely due to mild innate immune 275
response in Huh7 cells. It is evident from Fig 2C (lane 3) that PKR was over-expressed and 276
phosphorylated in cells lacking ANDV infection. However, the over-expressed PKR was poorly 277
phosphorylated in cells containing replicating ANDV (Fig 2C, lane 4), consistent with similar 278
observations from panels A and B. 279
To test the sensitivity of ANDV for PKR antiviral effects, we transfected Huh7 cells with 280
PKR expression plasmid 24 hours before or after ANDV infection. Virus replication was 281
monitored in cells at 48 and 72 hours post-infection by quantitative estimation of viral S-segment 282
RNA, using real time PCR. As shown in Fig. 2D, the prior expression of PKR inhibited ANDV 283
replication by ~ 75% (Fig 2D). In comparison, the later expression of PKR did not impact 284
ANDV replication (Fig. 2E). These results suggest that ANDV likely does not inactivate the 285
PKR that has been previously activated. 286
ANDV NP inhibits PKR autophosphorylation to prevent PKR induced translation 287
shutdown in cells. 288
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We next asked whether ANDV NP inhibits PKR autophosphorylation, especially due to its 289
high expression throughout the ANDV replication cycle. As lentivirus infections are known to 290
induce innate immune response, promote the expression and activation of PKR in cells, we asked 291
whether NP expression through lentivirus delivery system will inhibit the PKR-eIF2α pathway in 292
lentivirus infected cells. To test this hypothesis, HUVECs were infected with lentivirus at 293
increasing MOI, expressing either NP or EGFP as negative control. Cells were harvested 48 294
hours post-infection and activation of PKR-eIF2α pathway was examined by western blot 295
analysis. It was observed that lentiviral vectors expressing either EGFP or NP induced PKR 296
expression in a dose dependent manner (Fig. 3A). The infection with EGFP expressing lentivirus 297
activated the PKR-eIF2α pathway, evident from remarkable phosphorylation of both the PKR 298
and eIF2α (Fig. 3A, lanes 2-4). In comparison, the activation of PKR-eIF2α pathway was 299
significantly inhibited in cells infected with lentivirus expressing NP, evident from significant 300
reduction in the phosphorylation of both PKR and eIF2α (Fig. 3A, compare lane 7 with lane 4). 301
It has been previously reported that cytoplasmic tail domain of hantavirus glycoprotein Gn 302
regulates the early interferon response during virus infection (12). To determine whether Gn tail 303
domain also regulates the PKR activation similar to NP, HUVECs were infected with lentivirus 304
expressing either Gn tail domain or NP. An examination of cell lysates by western blot analysis 305
revealed that expression of Gn tail domain induced PKR expression and activation of PKR-eIF2α 306
pathway (Fig. 3B, lane 2). Consistent with the observations from Fig. 3A, the lentivirus infection 307
expressing NP induced PKR expression in a dose dependent manner, but failed to activate the 308
PKR-eIF2α pathway evident from negligible phosphorylation of both PKR and eIF2α (Fig. 3B, 309
compare lane 6 with lane 2). Based on the results from Fig. 3A and B, we envisioned that 310
lentivirus infection expressing EGFP in HUVECs will inhibit the host translation machinery 311
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whereas NP expression will rescue such translation shutdown. To test this hypothesis, HUVECs 312
were infected with increasing amount of lentivirus expressing either EGFP or NP. Forty-eight 313
hours post-infection, cells were chased for de novo protein synthesis by metabolic labeling, using 314
35S methionine and cysteine (Fig. 3C). As expected, the inhibition of de novo protein synthesis in 315
a dose dependent manner was observed in cells infected with lentivirus expressing EGFP (Fig. 316
3C). In comparison, the lentivirus infection expressing NP did not affect the rate of host protein 317
synthesis. These observations demonstrate that activation of PKR-eIF2α pathway by lentivirus 318
infection induces host translation shutoff, whereas inhibition of PKR activation by NP 319
expression rescues cells from such translation shutdown. Taken together, these studies 320
demonstrate that NP is a PKR inhibitor. 321
ANDV NP selectively inhibits PKR without impacting the activity of other eIF2α kinases. 322
There are four eIF2α kinases that phosphorylate eIF2α under different stress conditions or 323
stimuli and cause translation shutdown. These kinases include protein kinase R (PKR), PKR-like 324
endoplasmic reticulum kinase (PERK), heme regulated inhibitor (HRI) and general control non-325
derepressible-2 (GCN2) (33). PKR is activated by dsRNA, mostly during viral infection. HRI is 326
activated by heme deficiency, aimed to prevent the synthesis of globin peptides in response of 327
elevated heme levels. HRI is also activated by oxidative stress. PERK is activated by unfolded 328
protein response, triggered by the accumulation of misfolded proteins in the ER. GCN2 is 329
activated by amino acid starvation. Viral infections can induce various stress responses in the 330
host cell by the production of dsRNA intermediates or by competition with cellular translation 331
machinery and other resources for protein synthesis or by the alteration of cellular metabolism 332
(34). Since these diverse virus induced stress conditions may activate multiple eIF2α kinases, we 333
asked whether ANDV NP can also antagonize other eIF2α kinases and rescue cells from the 334
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induction of translation shutdown. We generated HEK293T stable cell lines constitutively 335
expressing either EGFP or NP from ANDV, SNV or HTNV. These cells were transfected with 336
poly I:C or stimulated with either DTT or sodium arsenite or subjected to amino acid starvation, 337
to activate PKR, PERK, HRI or GCN2, respectively (33), as mentioned in the legends of Fig. 4. 338
Cell lysates were examined for eIF2α phosphorylation using western blot analysis. It is evident 339
from Fig. 4A that poly I:C transfection activated PKR in control cells expressing EGFP, 340
observed by significant phosphorylation of both PKR and eIF2α (Compare lane 4 with lane 1 in 341
Fig. 4A). However, the cells expressing ANDV NP, SNV NP or HTNV NP comparatively 342
resisted the poly I:C induced phosphorylation of both PKR and eIF2α (Fig. 4A). This is 343
consistent with similar observations from Fig. 3. Interestingly, the stimuli activating PERK 344
(panel B), HRI (panel C) or GCN2 (panel D) equally induced the phosphorylation of eIF2α in 345
both EGFP and NP expressing cells, suggesting that NP likely does not inhibit the activation of 346
these kinases. Based on these observations it is likely that NP does not rescue cells from 347
translation shutdown induced by the stimuli activating PERK, HRI and GCN2. We used a 348
luciferase reporter assay to test this hypothesis. Firefly luciferase has a short half-life about 2 349
hours, making it suitable to monitor the rate of de novo protein synthesis in cells (35). The 350
HEK293T stable cell lines constitutively expressing either EGFP or NP were transfected with 351
pGL3-Fluc vector, and subjected to different treatments ten hours post-transfection for the 352
activation of PKR, PERK, HRI and GCN2, as described above. Cells lysates were examined for 353
luciferase activity using a luciferase assay kit (promega), following manufacturer’s instructions. 354
Luciferase activity in each group was normalized to untreated control (Fig. 4, Panels E-H). As 355
shown in Fig. 4E, the poly I:C treatment reduced luciferase activity by fifty percent in EGFP 356
expressing control cells, indicating translation shutdown. In comparison a marginal decrease in 357
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luciferase activity was observed in cells stably expressing ANDV NP, suggesting that NP 358
counteracts the PKR induced translation shutdown. The results together from Fig. 4A and 4E 359
suggest that NP expression inhibits PKR activation and rescues cells from PKR induced host 360
translation shutdown. However, the stimuli activating PERK (panel F), HRI (panel G) and GCN2 361
(panel H) equally reduced the luciferase activity in both EGFP and NP expressing HEK293T 362
stable cell lines, suggesting that NP expression does not rescue cells from translation shutdown 363
induced by PERK, HRI or GCN2. 364
Inhibition of PKR and eIF2α phosphorylation by NP does not require RNA sequestration 365
or recruitment of cellular phosphatases. 366
Viruses have evolved multiple strategies to antagonize PKR mediated antiviral responses. 367
Previous studies have revealed that viruses either express a decoy dsRNA or promote PKR 368
degradation or hide viral dsRNA or express pseudo-substrates or inhibit PKR dimerization or 369
activate PKR inhibitor P58IPK or directly dephosphorylate PKR or eIF2α to overcome PKR 370
mediated antiviral responses (16-18). High-level PKR expression during ANDV infection and 371
similar PKR induction in cells infected with lentivirus expressing either EGFP or NP suggest that 372
NP likely does not target PKR for degradation (Fig. 2 and Fig. 3). The requirement of dsRNA for 373
PKR autophosphorylation and NP being an RNA binding protein (22), led to the hypothesis that 374
sequestration of dsRNA by NP might result in the inhibition of PKR autophosphorylation. To 375
test this possibility, we deleted the RNA binding domain in NP and examined the potential of 376
resulting deletion mutant to inhibit the phosphorylation of PKR in cell culture. The RNA binding 377
domain of NP has been previously mapped to the region from 175-230 amino acids (Fig. 5A) 378
(22). HUVECs were infected with lentivirus expressing either EGFP or wild type NP or NP 379
mutant lacking the RNA binding domain. Forty-eight hours post-infection cells were harvested 380
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and PKR expression and activation was examined by western blot analysis. As shown in Fig. 5B, 381
similar levels of PKR induction were observed in HUVECs by the lentivirus infection expressing 382
either EGFP or wild type or mutant NP. However, unlike EGFP both the wild type and mutant 383
NP equally inhibited the phosphorylation of both PKR and eIF2α, suggesting that RNA binding 384
activity of NP likely does not play a role in the inhibition of PKR phosphorylation. To further 385
verify this observation, HEK293T cells stably expressing either EGFP or wild type or mutant NP 386
were transfected with poly I:C and cells were harvested at different time points post-transfection. 387
An examination by western blot analysis revealed that poly I:C treatment triggered the PKR 388
phosphorylation as early as one hour post-transfection in EGFP expressing control cells, which 389
was inhibited by the expression of both wild type and mutant NP (Fig. 5C). The effect of poly 390
I:C on PKR activation was time dependent, it stimulated more PKR phosphorylation at 2.5 hours 391
post-transfection (Fig. 5C), possibly due to better transfection efficiency of poly I:C at longer 392
incubation time. However, the activation of PKR was still equally inhibited by both wild-type 393
and mutant NP, suggesting the RNA sequestration is likely not the mechanism by which NP 394
inhibits PKR activation. To rule out the possibility that sequestration of poly I:C by NP inhibited 395
the PKR phosphorylation in this assay, HEK293T cell lysates expressing either wild type or 396
mutant NP or EGFP were incubated with poly I:C agarose beads. An examination of washed 397
beads by western blot analysis revealed that unlike PKR the wild type or mutant NP or EGFP did 398
not bind to poly I:C, demonstrating that possible sequestration of poly I:C by NP is not involved 399
in the inhibition PKR phosphorylation (Fig 5D). 400
To delineate the mechanism for the inhibition of PKR signaling by NP, we asked whether 401
NP recruits a phosphatas to dephosphorylate the activated forms of PKR and or eIF2α (36-38). 402
To test this hypothesis, HEK293T cells were first transfected with increasing amount of poly I:C 403
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for 3 hours to allow the phosphorylation of PKR and eIF2α, followed by transfection with 404
plasmids expressing either EGFP or wild type NP or NP mutant lacking the RNA binding 405
domain. Cells were harvested at 27 hours post poly I:C treatment and phosphorylation of both 406
PKR and eIF2α was monitored. As evident from Fig. 5E, transfection with poly I:C did not 407
affect the intrinsic steady state levels but induced the phosphorylation of both PKR and eIF2α in 408
a dose dependent manner, although the effect was more pronounced with PKR. Interestingly, the 409
expression of wild type or mutant NP did not impact the phosphorylation status of pre-410
phosphorylated PKR or eIF2α (Fig. 5E). This observation clearly demonstrates that inhibition of 411
PKR and eIF2α phosphorylation by NP-mediated mechanism does not involve the recruitment of 412
a possible phosphates. 413
Nucleocapsid protein inhibits PKR dimerization. 414
The dimerization induced auto-phosphorylation of PKR is a critical step in its activation 415
process. PKR has two dsRNA binding domains (RBDs) at the N-terminus and one kinase 416
domain (KD) at the C-terminus (39). The intra-molecular interaction between RBD and KD 417
renders KD in an inactive state (40, 41). The binding of dsRNA or PKR activating protein 418
(PACT) to the RBDs induces conformational change in PKR that relieves the intra-molecular 419
inhibition and promotes PKR dimerization by direct interaction between dimerization domains 420
(42). The dimerization triggers PKR auto-phosphorylation and renders the enzyme in fully active 421
form (43-45). We used immunoprecipitation approach to determine whether NP interferes in 422
PKR dimerization. HEK293T cells were co-transfected with plasmids expressing either wild 423
type or mutant NP or EGFP along with two additional plasmids, one expressing Myc tagged 424
PKR and another expressing FLAG tagged PKR. Cells were lysed at 48 hours post-transfection. 425
To promote the PKR dimerizaton, one third of the cell lysate was treated with poly I:C for 30 426
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minutes at 30°C before the lysates were subjected to immunoprecipiation using anti-FLAG 427
antibody. The immunoprecipiated material was examined by western blot analysis using anti-428
Myc tag antibody. It is evident from Fig. 6A that Myc and FLAG tagged PKR molecules in the 429
EGFP control experiment underwent dimerization, which was significantly promoted by poly I:C 430
treatment (compare lane 3 with lane 4). Interestingly, the co-expression of His-tagged NP or NP 431
mutant dramatically inhibited the interaction between Myc and FLAG tagged PKR molecules 432
(compare lanes 4 with lanes 8 and 12). This result demonstrates that NP inhibits PKR 433
dimerization. Since both wild type and mutant NP do not bind to poly I:C (Fig 5D), the possible 434
sequestration of poly I:C by NP is not likely the mechanism to inhibit PKR dimerization. 435
Moreover, it is evident from Fig 6A (lanes 7,8,11 and 12) that FLAG-PKR does not bind to His-436
NP. 437
Since hepatitis C virus NS5A protein has been reported to inhibit PKR dimerization through 438
binding to the PKR dimerization site (46, 47), we further confirmed that NP does not bind to 439
PKR. We co-transfected HEK293T cells with Myc tagged PKR and FLAG tagged NP. Cell 440
lysates were immunoprecipitated with either anti-FLAG tag antibody of IgG as negative control. 441
The immunoprecipitated material was examined by western blot analysis using anti-Myc tag 442
antibody. It is evident from Fig. 6B that NP does not bind to PKR. To further confirm this 443
observation, cell lysates were immunoprecipitated with either anti-Myc tag antibody or IgG. 444
Immunoprecipitated material was again examined by western blot analysis using anti-His tag 445
antibody. This reverse immunoprecipitation experiment (Fig. 6C) further demonstrates that NP 446
does not bind to PKR. 447
Discussion 448
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Modulation of innate immune response is a common strategy employed by viruses to survive 449
in infected hosts. Pathogenic hantaviruses such as ANDV, SNV and NY-1 virus achieve this goal 450
with the assistance of viral encoded glycoprotein to inhibit the type I interferon response during 451
early stages of viral infection (10, 12, 25, 48). However, the inhibition of early interferon 452
response does not seem to be sufficient for pathogenic hantaviruses to be human pathogens. This 453
is supported by the observations that Tula hantavirus (TULV) inhibits IFNβ production without 454
causing a serious human disease (49-52). Thus, the virulence factors for hantaviruses remain 455
incompletely defined. It is also well known that all hantaviruses strongly induce the expression 456
of both interferon and ISGs during the later stages of virus infection, which does not have effect 457
on the virus replication (11). Moreover it has been known that establishment of antiviral state 458
initiated by the transient shutdown of host translation machinery is not observed in hantavirus 459
infected cells (53). Consistent with these known findings, we observed that ANDV infection in 460
HUVECs induced PKR over-expression, which failed to shutdown the host translation 461
machinery and remained ineffective in combating the virus replication (Fig. 1). Further studies 462
demonstrated that although PKR was over-expressed during hantavirus infection, the over-463
expressed PKR was not activated due to the lack of autophosphorylation (Fig. 2A). As a result 464
the activity of downstream target eIF2α was not impaired, evident from lack of translation 465
shutdown in virus infected cells (Fig. 1C). Not only did ANDV infection inhibit the activation of 466
endogenous PKR but the over-expressed PKR from a transfected plasmid was also inhibited in 467
virus infected cells (Fig. 2C). The studies reported in this manuscript showed for the first time 468
that NP is a PKR inhibitor (Fig. 3). NP interferes in PKR dimerization, prerequisite for PKR 469
autophosphorylation and activation. A similar strategy is used by influenza virus and hepatitis C 470
virus (HCV) NS5A protein to mitigate the PKR antiviral responses (46, 54, 55). The mechanism 471
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by which NP inhibits PKR dimerization is unknown. We demonstrated that NP does not bind to 472
PKR (Fig. 6B and C), ruling out the possible interference of NP-PKR interaction in PKR 473
dimerization. Moreover, the possible recruitment of cellular phosphatases to dephosphorylate 474
PKR or eIF2α was also ruled out (Fig. 5E). In addition, the binding of PKR to dsRNA triggers 475
PKR dimerization and autophosphorylation. However, NP mutant lacking the RNA binding 476
domain was able to inhibit PKR similar to wild type NP, demonstrating that possible 477
sequestration of dsRNA by NP is not the mechanism to inhibit PKR dimerization. Viruses have 478
evolved numerous strategies to inhibit PKR antiviral response (16-18). However, PKR inhibition 479
by members of the Bunyaviridae family has not been extensively studied. The only reported 480
Bunyavirus inhibiting PKR is the Rift Valley Fever Virus (RVFV) who’s NSs protein has been 481
reported to promote PKR degradation (56, 57). However, the over-expression of PKR during 482
ANDV infection suggests that unlike RVFV, the hantavirus infection does lead to PKR 483
degradation. Thus, the actual mechanism for the inhibition of PKR dimerization by NP remains a 484
mystery. Nonetheless, the studies carried out in this manuscript demonstrate NP as a new 485
virulence factor for hantaviruses. These studies also shed light on the mechanism of hantavirus 486
resistance to host interferon response during later stages of infection, and solve the mystery of 487
lack of translation shutdown in the hantavirus infected cells. 488
Hantavirus NP is a multifunctional protein, primarily involved in the packaging of viral 489
genomic RNA in viral nucleocapsids. However, our recent studies have shown that NP has a role 490
in cap snatching mechanism of transcription initiation by viral RdRp (7). In addition, NP also 491
facilitates mRNA translation without the requirement of eIF4F cap binding complex (9, 58). Our 492
previous studies showed that NP binds to both the mRNA 5' cap and viral mRNA 5' UTR. In 493
addition, NP also binds to the 40S ribosomal subunit by direct interaction with the ribosomal 494
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protein S19 (RPS19), located at the head region of the 40S ribosomal subunit. Our previous 495
studies suggest that NP associated ribosomes are preferentially loaded on viral mRNA 5' UTR 496
thereby favoring their translation in the host cell cytoplasm where cellular transcripts are 497
competing for the same translation machinery (9, 59). However, both the canonical host 498
translation mechanism and NP-mediated translation strategy are dependent upon the eIF2α and 499
thus sensitive to PKR induced translation shutdown. This is indirectly supported by the fact that 500
over-expression and activation of PKR in transfected cells prior to ANDV infection significantly 501
impaired virus replication (Fig. 2D). The sensitivity of hantavirus replication to PKR antiviral 502
response has likely imposed a selective evolutionary pressure on hantaviruses to evade PKR 503
antiviral response for survival. Our studies suggest that inhibition of PKR antiviral response by 504
NP has likely helped hantavirus to exist and survive in the infected hosts over the course of 505
evolution. 506
PKR is a multifunctional protein, which not only inhibits host translation machinery but 507
also plays critical role in numerous signal transduction pathways. For example PKR plays a 508
positive feedback role in IFN signaling pathway and its inhibition attenuates the induction of 509
genes normally stimulated by IFN (60). PKR positively regulates the induction of IFNβ in 510
responses to viral infection via NF-κB and IRF1 (61). PKR also function as cytosolic dsRNA 511
sensor and activates NF-κB by activating NIK and IKK kinases (62). Since NF-κB broadly 512
regulates the induction of antiviral genes and inflammatory cytokines, its activation favors the 513
quick establishment of antiviral state, which is not observed in hantavirus infected cells (63). 514
Moreover, PKR has been implicated to play a role in apoptotic pathways. Over-expression of 515
PKR sensitizes cells to apoptosis induced by dsRNA, TNFα and virus infection (64). Apoptosis 516
is the ultimate cellular measure in controlling virus replication and spread (65). However, 517
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hantavirus infected cells do not undergo apoptosis (66), likely due to the inhibition of PKR by 518
NP. PKR also plays a key role in the formation of antiviral stress granules (avSGs), which serves 519
as platforms for sensing the non-self RNAs by RIG-I like receptors (67). These reported findings 520
demonstrate that PKR is a multifunctional antiviral host factor. Being a PKR inhibitor, NP would 521
likely antagonize the diverse PKR antiviral responses in virus infected cells and create a 522
supportive environment for virus replication. Taken together, our results illustrate that 523
multifunctional nature of viral proteins help viruses to carry small size genomes and still survive 524
in hosts having multifaceted antiviral defense. 525
Acknowledgement 526
We would like to thank Joseph Prescott from NIH/NIAID for providing ANDV stocks used in 527
this work. This work was supported by NIH grants RO1 AI095236-01 and 1R21 AI097355-01. 528
Zekun Wang did all the experiments, wrote the first draft of this manuscript and provided all 529
figures published here. All authors red the manuscript before publication. 530
531
Figure Legends 532
Fig. 1 ANDV stimulated PKR expression but did not impact the host protein synthesis. 533
(A) HUVECs were infected with ANDV at an MOI of 0.5, cells were harvested at different time 534
points post-infection. PKR and NP expression were analyzed by western blot. GAPDH was used 535
as loading control. (B) HUVECs were infected with ANDV and harvested as described in Fig. 536
1A. PKR mRNA levels were examined by real time PCR, normalized to β-actin mRNA levels 537
and shown as fold change relative to mock cells. Results from three independent experiments 538
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were used to calculate standard deviation, shown as error bars. The significance (*) was 539
calculated by t-test. (C) HUVECs were infected with ANDV as described above. Cellular de 540
novo protein synthesis was monitored by 35S-methionine/cysteine incorporation at different time 541
points post-infection. Protein samples were separated by SDS-PAGE and visualized by 542
coomassie staining (right panel) and autoradiography (left panel). (D) The bands intensities of 543
the autoradiogram were quantified by Image-Pro Plus software, averaged and normalized to the 544
mock. Results from three independent experiments were used to calculate the standard deviation 545
and shown as error bars. 546
Fig. 2 ANDV infection interfered with PKR phosphorylation. 547
(A) HUVECs in six well plates were infected with ANDV and cells were harvested as described 548
in Fig. 1A. In addition, HUVECs were treated with IFNα (1000 U/ml) for 16 hours. Following 549
IFNα treatment, cells were either mock transfected or transfected with poly I:C (200 ng/ml) for 550
additional 2 hours before harvesting. Cell lysates were examined for the expression of PKR, 551
eIF2α and their phosphorylated forms by western blot analysis using appropriate antibodies. 552
GAPDH was used as loading control. (B) HUVECs were infected with ANDV at an MOI of 0.5 553
or lentivirus at an MOI of 30 to stimulate comparable expression level of PKR. Cells were 554
harvested at 48 and 72 hours post-infection. The levels of PKR, p-PKR, eIF2α and p-eIF2α, 555
ANDV NP, EGFP and GAPDH in cell lysates were detected by western blot using appropriate 556
antibodies. (C) Huh7 cells were infected with Andes virus at an MOI of 1, followed by 557
transfection with either empty vector or a plasmid expressing N-terminally Myc tagged PKR 24 558
hours post-infection. Cells were harvested at 48 hours post-infection. The levels of PKR, p-PKR, 559
NP and GAPDH were detected using appropriate antibodies. (D) Huh7 cells in six well plates 560
were transfected with PKR expression plasmid, followed by ANDV infection 24 hours post-561
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transfection at an MOI of 0.2. Cells were harvested at 1, 48 and 72 hours post-infection and viral 562
S-segment RNA was quantified by real time PCR. The RNA levels were normalized to 1 hour 563
post-infection. Data from three independent experiments was averaged and used to calculate the 564
standard deviation, shown as error bars. (E) Huh7 cells were infected with ANDV at an MOI of 565
0.2. Cells were transfected with PKR expressing plasmid 24 hours post-infection and harvested 566
72 hours post-infection. Viral S-segment RNA was quantified using the same method as 567
mentioned in panel D. 568
Fig. 3 ANDV NP inhibits PKR phosphorylation. 569
(A) HUVECs were infected with lentivirus expressing either EGFP (Lenti-EGFP) or NP (Lenti-570
NP) at an increasing MOI, ranging from 10 to 30. Cells were harvested at 48 hours post-infection 571
and the expression of PKR, p-PKR, eIF2α, p-eIF2α, EGFP and NP were detected using 572
corresponding antibodies. Lysate from mock-infected cells was used as negative control. The 573
levels of β-actin were used as loading control. (B) HUVECs were infected with lentivirus 574
expressing the cytoplasmic tail domain of glycoprotein Gn (Lenti-Gntail) at an MOI of 40, or 575
infected with Lenti-NP at an increasing MOI ranging from 10 to 40. Cells were harvested at 48 576
hours post-infection and cell lysates were examined by western blot analysis as mentioned in 577
panel A. (C) HUVECs were infected with lentivirus (Lenti-EGFP or Lenti-NP) at increasing 578
MOI as mentioned in Fig. 3A, followed by metabolic labeling with 35S-methionine/cysteine at 48 579
hours post-infection. Cells were lysed and protein samples were separated by SDS-PAGE and 580
visualized by coomassie staining (right panel). The same gel was dried and exposed to x-ray film 581
(left panel). (D) The band intensities of the autoradiogram were quantified by Image-Pro Plus 582
software and normalized to that of mock control. Results from three independent experiments 583
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were used to calculate standard deviation and shown as error bars. The significance (*) was 584
calculated by t-test. 585
Fig. 4 ANDV NP selectively inhibited PKR. 586
(A) HEK293T cells stably expressing either EGFP or ANDV-NP, SNV-NP, HTNV-NP were 587
transfected with poly I:C (400 ng/ml) for 2.5 hours prior to lysis. Cell lysates were examined for 588
the expression of PKR, p-PKR, eIF2α, p-eIF2α, EGFP, NP and β-actin by western blot analysis 589
using appropriate antibodies. (B, C, D) The HEK293T stable cell lines mentioned above were 590
treated with 2 mM DTT (panel B) or 0.1 µM arsenite (panel C) for two hours before lysis. In 591
panel D, the cells were cultured in starvation medium for 24 hour before lysis. The expression of 592
eIF2α, p-eIF2α, EGFP, NP and β-actin were detected by western blot. (E, F, G, H) The 593
HEK293T stable cell lines mentioned above were transfected with 100 ng pGL3-Fluc plasmid. 594
Ten hours post-transfection, cells were transfected with 400 ng/ml poly I:C (panel E) or treated 595
with 2 mM DTT (panel F), or treated with 0.1 µM arsenite (panel G) for eight hours before lysis 596
or subjected to starvation medium for 24 hours before lysis (panel H). The luciferase activities 597
were measured and normalized to that of the un-treatment control in the same group. Results 598
from three independent experiments were used to calculate error bars. Note: * represents the 599
significant difference calculated by t-test and NS represents not significant. 600
Fig. 5 The RNA binding activity of NP and cellular phosphatases are not involved in NP-601
mediated PKR inhibition. 602
(A) Schematic representation of wild type ANDV NP and NP (Δ175-230) mutant. (B) HUVECs 603
were infected with lentivirus expressing either EGFP or NP or NP (Δ175-230) mutant at an MOI 604
of 30. Cells were harvested at 48 hours post-infection and expression of PKR, p-PKR, eIF2α, p-605
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eIF2α, EGFP, wild type NP and NP (Δ175-230) mutant were examined by western blot analysis. 606
Cell lysate from uninfected cells was use as mock control. (C) HEK293T stable cell lines 607
expressing either EGFP or wild type NP or NP (Δ175-230) mutant were transfected with 400 608
ng/ml poly I:C for one or 2.5 hours before harvesting. Cell lysates were examined for the 609
expression of PKR, p-PKR, EGFP, NP, NP (Δ 175-230) and β-actin by western blot analysis 610
using appropriate antibodies. (D) The poly I:C pull down assays were carried out using a 611
standard protocol (68). Briefly, HEK293T cells stably expressing either EGFP or NP or NP 612
(Δ175-230) were lysed with lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 613
mM EDTA). The resulting cell ysates were incubated with poly I:C-coated beads (Sigma) for 1 614
hour at 4°C with gentle agitation. The beads were extensively washed with lysis buffer and 615
bound proteins were eluted by boiling with 1× SDS loading buffer. The eluted proteins were 616
examined by western blot using appropriate antibodies. (E) HEK293T cells were transfected 617
with poly I:C at increasing concentration ranging from 100 to 400 ng/ml for 3 hours, followed by 618
transfection with plasmids expressing either EGFP or NP or NP (Δ175-230) mutant. Cells were 619
lysed at 27 hours post poly I:C transfection and protein samples were examined by western blot 620
analysis to monitor the expression levels of PKR, p-PKR, NP, NP (Δ175-230) mutant, EGFP and 621
β-actin, using appropriate antibodies. 622
Fig. 6 Andes virus NP inhibits PKR dimerization. 623
(A) HEK293T cells were cotransfecetd with plasmids expressing either EGFP or wild type or 624
mutant His-tagged NP along with two additional plasmids, one expressing Myc tagged PKR and 625
another expressing FLAG tagged PKR. Cells were lysed at 48 hours post-transfection and one 626
third of the cell lysate was treated with poly I:C (1 µg/ml) for 30 min. Cell lysates were 627
immunoprecipitated with either IgG or anti-FLAG antibody and immunoprecipitated material 628
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was examined by western blot analysis using either anti-Myc tagged antibody to detect Myc 629
tagged PKR or anti-FLAG antibody to detect FLAG tagged PKR or anti-NP antibody to detect 630
His-tagged NP or anti-GFP to detect GFP or anti-GAPDH antibody to detect GAPDH. (B) 631
HEK293T cells were co-transfected with plasmids expressing Myc tagged PKR and FLAG 632
tagged NP. Forty-eight hours post-transfection, cells were lysed with 0.5% NP-40 lysis buffer. 633
Cell lysates were immunoprecipitated with either anti-FLAG tag antibody or IgG. The 634
immunoprecipitated material was examined by western blot analysis using either anti-Myc tag 635
antibody (top panel) or anti-FLAG tag antibody (middle panel). Bottom panel shows IgG light 636
chain. (C) The experiment in panel C was performed the same way as panel B, except 637
immunoprecipitation was carried out using anti-Myc tag antibody and western blot analysis was 638
carried out using anti-His tag antibody. 639
640
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